The use of unmodified stroma-free hemoglobin has been proposed as a resuscitating solution for many years. However, there are two major intrinsic problems considered to hinder use of unmodified solution. The first is rapid renal excretion or clearance due to the dissociation of the hemoglobin tetramer. The second is worsening of the hemoglobin's ability to unload oxygen to the tissues that occurs due to the isolation of hemoglobin from the red blood cell and its components.
At least a decade ago it was stated regarding the fragility of hemoglobin that the slightest disturbance in hemoglobin structure is immediately reflected in the ability of its divalent ion to combine reversibly with molecular oxygen while denaturation of the protein results in complete loss of the property of oxygenation.
Nonetheless, several approaches have attempted to address both of the above-referred to problems of rapid renal clearance and high oxygen affinity but almost invariably existant modifications have solved one but not both of these issues.
In the former case, three attempts to crosslink hemoglobin to improve its intravascular retention are detailed in U.S. Pat. Nos. 4,061,736 and 4,001,401 to Morris et al, and 3,925,344 to Mazur et al. These types of reactions are universally plagued with the problem that once the hemoglobin is modified by these reactions, the modified or crosslinked product does not effectively unload the oxygen carried.
The problem of improving oxygen unloading has been addressed by a number of reactions that do not solve the renal loss problem (see, for example, a review paper entitled "Oxygen Equilibrium and Structural Studies of Amidinated Human Hemoglobin", by B. Horowitz and A. Mazur, Blood Substitutes and Plasma Expanders, Alan R. Liss, Inc., 149-165 (1978)).
A published work, also referred to in Table 1 of the Horowitz and Mazur review, by Benesch, et al, Biochem. Biophys. Res. Comm., Vol. 63, 1123-1129 (1975) is considered an advance in the attempted solution to both of the major problems referred to above. However, the crosslinking agent used, 2-nor-2-formyl-pyridoxal-5-phosphate, is not commercially available and is difficult to obtain even for experimental purposes.